Abstract Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for many types of hematologic malignancies. However, graft-versus-host disease (GVHD) mediated by alloreactive T cells remains the most common cause of nonrelapse-related morbidity and mortality after allo-HSCT. Although T cell depletion and other strategies of reducing T cell alloreactivity are effective in the control of GVHD, they are often associated with increased risks of tumor recurrence. Thus, it is compelling to develop strategies for enhancing anti-tumor effect without GVHD. Studies have shown that donor natural killer (NK) cell alloreactivity can exert anti-tumor effect while protecting patients against GVHD. However, it is not clear whether these donor-derived alloreactive NK cells can persist to become long-lived memory cells and protect hosts from tumor recurrence. Although immunological memory has been thought to be a hallmark of adaptive immunity, recent advance has suggested that specific subsets of NK cells can develop into long-lived and specific memory cells in models of hapten-induced contact hypersensitivity and viral infections. In a murine model of allogeneic transplantation, we showed in the preliminary studies that alloreactive NK cells can persist for a long time, possess the properties of memory cells and protect against a secondary tumor challenge. The objective of this application is to understand the mechanisms underlying the formation of alloreactive memory NK cells. We hypothesize that multiple signals are required for the formation of alloreactive NK cell memory. We will test this hypothesis with four specific aims and pursue critical elements required for the formation of alloreactive memory NK cells. By providing molecular and cellular basis for the formation of alloreactive NK cell memory, these studies will have profound implications in the development of new therapeutic strategies for cancer prevention and therapy in the setting of allogeneic stem cell transplantation. Furthermore, the principles learned here can be applied to the NK cell memory field in general for the treatment of other diseases such as pathogenic infections.